RAS mutations are frequent in human cancer, especially in pancreatic, colorectal and non-small-cell lung cancers (NSCLCs). Inhibition of the RAS oncoproteins has proven difficult, and attempts to target downstream effectors have been hampered by the activation of compensatory resistance mechanisms. It is also well established that KRAS-mutant tumors are insensitive to inhibition of upstream growth factor receptor signaling. Thus, epidermal growth factor receptor antibody therapy is only effective in KRAS wild-type colon cancers. Consistently, inhibition of SHP2 (also known as PTPN11), which links receptor tyrosine kinase signaling to the RAS-RAF-MEK-ERK pathway, was shown to be ineffective in KRAS-mutant or BRAF-mutant cancer cell lines. Our data also indicate that SHP2 inhibition in KRAS-mutant NSCLC cells under normal cell culture conditions has little effect. By contrast, SHP2 inhibition under growth factor-limiting conditions in vitro results in a senescence response. In vivo, inhibition of SHP2 in KRAS-mutant NSCLC also provokes a senescence response, which is exacerbated by MEK inhibition. Our data identify SHP2 inhibition as an unexpected vulnerability of KRAS-mutant NSCLC cells that remains undetected in cell culture and can be exploited therapeutically.

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Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress-response programs that counteract the inherent toxicity of such aberrant signaling. While inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of Protein Phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor suppressive resistance.

Precision oncology aims to distinguish which patients are eligible for a specific treatment in order to achieve the best possible outcome. In the last few years, genetic screens have shown their potential to find the new targets and drug combinations as well as predictive biomarkers for response and/or resistance to cancer treatment. In this review, we outline how precision oncology is changing over time and describe the different applications of genetic screens. Finally, we present some practical examples that describe the utility and the limitations of genetic screens in precision oncology.

Discovering biomarkers of drug response and finding powerful drug combinations can support the reuse of previously abandoned cancer drugs in the clinic. Indisulam is an abandoned drug that acts as a molecular glue, inducing degradation of splicing factor RBM39 through interaction with CRL4DCAF15. Here, we performed genetic and compound screens to uncover factors mediating indisulam sensitivity and resistance. First, a dropout CRISPR screen identified SRPK1 loss as a synthetic lethal interaction with indisulam that can be exploited therapeutically by the SRPK1 inhibitor SPHINX31. Moreover, a CRISPR resistance screen identified components of the degradation complex that mediate resistance to indisulam: DCAF15, DDA1, and CAND1. Last, we show that cancer cells readily acquire spontaneous resistance to indisulam. Upon acquiring indisulam resistance, pancreatic cancer (Panc10.05) cells still degrade RBM39 and are vulnerable to BCL-xL inhibition. The better understanding of the factors that influence the response to indisulam can assist rational reuse of this drug in the clinic.

Abbreviations BRAF, v-raf murine sarcoma viral oncogene homolog B1; CRISPR, clustered regularly interspaced short palindromic repeats; ERK, extracellular signal-regulated kinase; FDA, U.S. Food and Drug Administration; HCC, hepatocellular carcinoma; H&E, hematoxylin and eosin; KRAS, Kirsten rat sarcoma viral oncogene homolog; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian/mechanistic target of rapamycin; NSCLC, non-small-cell lung cancer; PROTAC, proteolysis targeting chimera; PTPN11, protein tyrosine phosphatase non-receptor type 11; qRT-PCR, quantitative real-time polymerase chain reaction; RTK, receptor tyrosine kinases; SHP2, Src homology 2 (SH2) domaincontaining tyrosine phosphatase-2; TNCB, triple negative breast cancer.

Mutant KRAS is present in over 90% of pancreatic as well as 30-40% of lung and colorectal cancers and is one of the most common oncogenic drivers. Despite decades of research and the recent emergence of isoform-specific KRASG12C-inhibitors, most mutant KRAS isoforms, including the ones frequently associated with pancreatic ductal adenocarcinoma (PDAC), cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive mechanisms leading to tumor recurrence or resistance. We report here on the combined inhibition of SHP2, a non-receptor tyrosine phosphatase upstream of KRAS, and ERK, a serine/threonine kinase and a key molecule downstream of KRAS in PDAC. This combination shows synergistic anticancer activity in vitro, superior disruption of the MAPK pathway, and significantly increased apoptosis induction compared to single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination. Concurrent inhibition of SHP2 and ERK induces significant tumor regression in multiple PDAC mouse models. Finally, we show evidence that 18F-FDG PET scans can be used to detect and predict early drug responses in animal models. Based on these compelling results, we will investigate this drug combination in a clinical trial (SHERPA, SHP2 and ERK inhibition in pancreatic cancer, NCT04916236), enrolling patients with KRAS-mutant PDAC.

IntroductionPancreatic ductal adenocarcinoma (PDAC) is a tumour with a fibroblastic stroma compartment that consists of pancreatic stellate cells (PSC) which play a complex role in supporting carcinogenesis, immunosuppression and therapy resistance. Therefore, the 5 year survival rate for PDAC patients remains below a disappointing 8%, stressing the need for new and more effective treatments. Recently, cold atmospheric plasma (CAP) has emerged as a potent treatment option for cancer. Although, CAP is being investigated for several years, the involvement of the immune system after CAP treatment remains poorly understood. The immunogenic cell death (ICD) concept describes that the killing of cancer cells leads to direct activation of the immune system through release of so-called ‘danger-associated molecular patterns’. ICD can be elicited by several physical means such as irradiation and photodynamic therapy, providing a rationale for the induction of ICD after CAP treatment. The aim of this study is to investigate the induction of a specific antitumoral immune response after CAP treatment in PDAC, in vitro.Material and methodsPhosphate-buffered saline (PBS) was treated with CAP, generated by the kINPenIND, and subsequently added to monocultures of both pancreatic cancer cell (PCC) lines and PSC lines. To evaluate the four most important hallmarks of ICD, being membrane exposure of calreticulin, secretion of ATP and release of HMGB1 and type I interferon, the treatment parameters were optimised (i.e. treatment time, gas flow and gap distance) to obtain 50% cell death. The cellular difference in sensitivity for CAP treatment was assessed through cytotoxic analysis. After attaining the optimal treatment parameters, we investigated the translocation of calreticulin onto the cell surface with flow cytometry. ATP secretion was investigated with a bioluminescence assay, while ELISA was used to monitor the release of HMGB1 and interferon type I in the supernatants.Results and discussionsOur data report a cytotoxic effect of CAP treatment in vitro on both PCC and PSC. In both PCC and PSC our results show a significant expression of calreticulin after CAP treatment, together with a significant release of ATP in PCC, but not in PSC. The evaluation of other ICD hallmarks after CAP treatment is currently ongoing.ConclusionWe strongly believe that CAP therapy can be a good alternative for the treatment of PDAC. However, the results warrant further in vivo validation to refine the involvement of the immune system after CAP treatment.